Organophosphorus (OP) compounds, which have been used as chemical warfare agents (CWAs), inhibit acetylcholinesterase (AChE) in neural synapses, glands, and neuromuscular junctions. This action results in an accumulation of excess acetylcholine which, at lethal levels, will lead to death from respiratory failure (Sidell, 1997). High level OP poisoning with CWAs can result in seizures from the excessive central hyperexcitability, leading to short-term incapacitation and long-term brain damage. CWA exposure has resulted in deficits in psychomotor function and memory, as evidenced in humans exposed to sarin in the Japanese terrorist attacks (Nishiwaki et al., 2001; Miyaki et al, 2005).
Chemotherapy for OP poisoning typically involves the administration of atropine, a muscarinic receptor antagonist, and an oxime (e.g., 2-PAM), an AChE reactivator (Dunn et al., 1997). While other approaches have been investigated for the treatment of OP poisoning, oxime therapy to rescue inhibited AChE still appears to be an essential therapeutic.
AChE reactivation by oximes occurs through nucleophilic attack on the OP moiety bound to the enzyme. The administration of an oxime must occur before the inhibited AChE has “aged”, a non-enzymatically mediated dealkylation reaction that renders the inhibited enzyme refractory to either spontaneous or oxime-mediated reactivation. The time course of this AChE aging is compound dependent, with a compound such as soman displaying very rapid aging. Therefore, for oxime therapy to be effective, it generally needs to be administered and delivered to the inhibited AChE quickly after CWA exposure, before appreciable proportions of the inhibited AChE ages.
A number of oximes have been synthesized and tested as AChE reactivators since the initial discovery of 2-PAM (Wilson and Ginsberg, 1955), including bis-pyridinium oximes containing at least one pyridine-2- or -4-aldoxime moiety (Worek et al., 1996; Kassa and Cabal, 1999; Chen et al., 2001; Kuca and Kassa, 2004; Kim et al., 2005; Kuca et al., 2005; Petroianu et al., 2006). Despite years of research, however, 2-PAM and obidoxime remain the most widely-used AChE reactivators (Sidell, 1997).
A significant limitation to the value of existing oximes is their lack of effectiveness in the brain. This limitation is due to the inability of large highly polar molecules to cross the blood-brain barrier. The existing oximes can allow OP poisoning victims to survive, by reactivating the phosphorylated AChE in the peripheral nervous system (e.g., in the diaphragm and lungs), while the brain AChE remains substantially inhibited for an extended period of time. A recent study used microdialysis and HPLC to detect 2-PAM in the brain of rats following intravenous administration of the oxime. However, the level found in the brain was only about 10% of that in the blood (Sakurada et al., 2003).
Due to the limited ability of 2-PAM to cross the blood brain barrier and reactivate brain AChE, a pro-drug form of 2-PAM, pro-PAM, was synthesized. Initial studies with pro-PAM suggested increased brain AChE activity (Clement, 1979); however, subsequent studies demonstrated that pro-PAM was more toxic than 2-PAM. Additionally, pro-PAM's ability to reactivate brain AChE was dependent on the rate at which the drug was transported to the CNS, with intravenous administration producing the only significant brain AChE reactivation (Kenley et al, 1982). Therefore, pro-PAM, requiring intravenous administration, is not practical for poisonings occurring on the field of battle. Additionally, a drug requiring metabolic activation in a tissue (i.e., brain) known for its low levels of drug metabolizing enzymes, is not ideal for rapid therapy against agents, such as soman, which result in rapid aging of phosphorylated AChE.
Reactivating OP-inhibited AChE in the brain is important, as OP compounds can have adverse effects on memory for years after a poisoning incident. Studies in laboratory animals have indicated that neurochemical aberrations persist well after the initial exposure to insecticidal OP's and well after the AChE activity has recovered to normal levels (Richardson and Chambers, 2003, 2004, 2005; Tang et al., 1999, 2003). Long-term memory deficits have likewise been reported in the victims of terrorist attacks with sarin in Japan (Nishiwaki et al., 2001; Miyaki et al., 2005). Such long-term effects on brain chemistry and on behavior, undoubtedly resulting from impacts of poisoning on the brain, would be expected to be exacerbated if the AChE inhibition were prolonged. Returning AChE activity to normal levels quickly, in order to rapidly return a subject's neurochemistry to normal, would be expected to reduce both short-term and long-term physiological and behavioral effects. Therefore, an antidote effective in the brain should be extremely valuable in not only survival of lethal dose exposures, but also returning a subject to normalcy as quickly as possible.